蛋黄中卵黄高磷蛋白的提取优化及其磷酸肽制备

Isolation of Egg Yolk Phosvitin and Preparation of Its Phosphopeptides

卵黄高磷蛋白是已知天然蛋白中含磷量最高的蛋白,经适宜蛋白酶水解后形成磷酸肽,该磷酸肽能够与Ca2+、Fe2+、Mg2+等二价金属离子结合形成可溶性络合物,抑制磷酸钙等非可溶性磷酸盐的形成,从而促进机体对钙、铁的吸收。本文主要探索了不同缓冲溶液、不同稀释程度、不同p H值等因素对卵黄高磷蛋白提取效果的影响,并在单因素实验的基础上采用正交试验,进一步优化了卵黄高磷蛋白的提取工艺。结果表明,应用10倍体积的0.05 M的p H 5.2的醋酸-醋酸钠溶液提取的卵黄高磷蛋白效果较好,每10 m L的卵黄可以提取约90mg的卵黄高磷蛋白。提取的卵黄高磷蛋白成品含磷量为7.2%,N/P比为3.9。经过进一步脱磷、酶解等处理制备了卵黄高磷蛋白磷酸肽,该磷酸肽能明显阻滞磷酸钙沉淀的形成,每100 mg能够结合9.1 mg的钙。本实验结果,为后续动物体内补钙功能实验及相关磷酸肽补钙制剂的研发奠定了基础。

Egg yolk phosvitin is the highest phosphorylated protein found in the nature,and is considered to be the best source for phosphopeptides production. Phosphopeptides can effectively bind calcium,iron and magnesium,and inhibit the formation of insoluble calcium phosphates or iron complexes. Phosphopeptides are developed as a new carrier to promote the body＇ s absorption of calcium,iron and magnesium. In this paper,several important technologic conditions of phosvitin preparation,including buffer solution,p H and dilution times were optimized by the single factor test and orthogonal experiment. The results showed that phosvitin can be well isolated with 10 volumes of 0. 05 M p H 5. 2 Na Ac buffer dilution. The yield of phosvitin was 90 mg from 10 m L of egg yolk,and its the phosphorus content was 7. 2%,the N / P ratio was 3. 9. Furthermore,the phosvitin product was dephosphoried with 0. 2 M Na OH for 2 h,and then hydrolyzed with trypsin for 4 h to get phosphopeptide. In the further experiment,the phosvitin phosphopeptide product was demonstrated to have a strong activity of blocking formation of calcium phosphate precipitation,and its calcium binding ability reached up to 9. 1%. Altogether,these results laid solid foundation for further in vivo studying the function of phosvitin phosphopeptide.